In tissues that do not require insulin for glucose uptake, the high systemi level of glucose that develops during diabetic hyperglycemia readily translates into high tissue levels of glucose. Some of this excess glucose is metabolized by the polyol pathway. Aldose reductase (AR), the first enzyme of this pathway, reduces glucose to the organic osmolyte sorbitol while sorbitol dehydrogenase (SDH), the second enzyme of the pathway, oxidizes sorbitol to fructose. The diabetes-enhanced flux of glucose through the polyol pathway has been implicated in the etiology of diabetic complications, including cataract, retinopathy, and neuropathy. Our studies have been aimed at defining the structure and regulation of AR and SDH so that new approaches to the control of this pathway may be made available in diabetic tissues. Many aldose reductase inhibitors (ARIs) have been shown to have broad substrate specificity and undesirable side effects. Emphasis on the structure/function properties of the AR enzyme will help in the refinement and design of future inhibitors. To this end, catalysis and inhibition of aldose reductase was examined following site-directed mutagenesis. In the rat, our mutagenesis studies indicated that tyrosine 48, histidine 110, and cysteine 298 are important residues in the active site and that tyrosine 48 is most likely the proton donor during substrate reduction. In addition, mutation of histidine 110, an active-site residue, decreased inhibitor effectiveness by up to 2000-fold, indicating that this residue is important in inhibitor binding. Mutations of human AR indicated that although phenylalanine 122 putatively binds ARIs, substitution of this amino acid ha little affect on the inhibitor-binding constant, Ki. Sorbitol dehydrogenase has been reported to be linked to cataract formation in nondiabetics. We have determined the gene structure, tissue distributio transcriptional initiation, and chromosomal localization of human SDH. In addition we have used SSCP analysis followed by sequence comparison to analyze congenital cataract patients with SDH deficiency. Through these studies, we hope to identify the gene defect in this family.